[Show abstract][Hide abstract] ABSTRACT: WNT signaling stimulates the self-renewal of many types of adult stem cells, including mammary stem cells (MaSCs), but mechanisms that limit this activity are poorly understood. Here, we demonstrate that SLIT2 restricts stem cell renewal by signaling through ROBO2 in a subset of basal cells to negatively regulate WNT signaling. The absence of SLIT/ROBO2 signaling leads to increased levels of nuclear β-catenin. Robo2 loss does not increase the number of stem cells; instead, stem cell renewal is enhanced in the absence of SLIT/ROBO2 signaling. This is due to repressed expression of p16(INK4a), which, in turn, delays MaSC senescence. Together, our studies support a model in which SLITs restrict the expansion of MaSCs by countering the activity of WNTs and limiting self-renewal.
[Show abstract][Hide abstract] ABSTRACT: SLIT expression is reduced in majority of cancers, establishing a correlation between SLIT and deregulated cell division. In the mammary gland, SLIT acts as a signaling molecule through ROBO receptors, ROBO1 and ROBO2. Preliminary data show that serial transplantation of Slit2-/-;Slit3-/- mammary gland tissue results in enhanced longevity. Moreover, we see the opposite effect in vitro, by treating mammospheres with SLIT2. Together these results suggest that SLIT functions as a non-renewal factor for mammary stem cells. Given these data, my project is to determine which receptor, ROBO1 or ROBO2, is responsible for SLIT signaling. Further, we have two cell types of interest, myoepithelial and luminal cells that generate the epithelial tree of the mammary gland. We enriched for Robo1-/- and +/+ mammary stem cells using FACS (Fluorescent Activated Cell Sorting) and then plated and cultured these cells in Matrigel. The cultures were treated with 1,000 ng/mL or 2,000 ng/mL of SLIT2. Preliminary results show that SLIT2 treatment of Robo1-/-mammary stem cells at either concentration, results in an increase in luminal cell fate. These data appear to rule out ROBO1 as the operative receptor since we observe an effect with Robo1-/- cells. Our next step will be to analyze Robo2-/-mammary stem cells in similar experiments, and also use qPCR along with immunohistochemistry to validate the observation that ROBO2 is the receptor that mediates SLIT/ROBO signaling.
[Show abstract][Hide abstract] ABSTRACT: Slit, Netrin, Ephrin, and Semaphorin's roles in development have expanded greatly in the past decade from their original characterization as axon guidance molecules (AGMs) to include roles as regulators of tissue morphogenesis and development in diverse organs. In the mammary gland, AGMs are important for maintaining normal cell proliferation and adhesion during development. The frequent dysregulation of AGM expression during tumorigenesis and tumor progression suggests that AGMs also play a crucial role as tumor suppressors and oncogenes in breast cancer. Moreover, these findings suggest that AGMs may be excellent targets for new breast cancer prognostic tests and more effective therapeutic strategies.
Full-text · Article · Sep 2011 · Journal of Mammary Gland Biology and Neoplasia
[Show abstract][Hide abstract] ABSTRACT: In the field of breast biology, there is a growing appreciation for the "gatekeeping function" of basal cells during development and disease processes yet mechanisms regulating the generation of these cells are poorly understood. Here, we report that the proliferation of basal cells is controlled by SLIT/ROBO1 signaling and that production of these cells regulates outgrowth of mammary branches. We identify the negative regulator TGF-β1 upstream of Robo1 and show that it induces Robo1 expression specifically in the basal layer, functioning together with SLIT2 to restrict branch formation. Loss of SLIT/ROBO1 signaling in this layer alone results in precocious branching due to a surplus of basal cells. SLIT2 limits basal cell proliferation by inhibiting canonical WNT signaling, increasing the cytoplasmic and membrane pools of β-catenin at the expense of its nuclear pool. Together, our studies provide mechanistic insight into how specification of basal cell number influences branching morphogenesis.
Full-text · Article · Jun 2011 · Developmental Cell
[Show abstract][Hide abstract] ABSTRACT: FOXA1, estrogen receptor alpha (ERalpha) and GATA3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERalpha and GATA3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERalpha expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike ERalpha and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERalpha expression, but not GATA3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERalpha-positive breast cancers, at least in part, through its control of ERalpha expression.
[Show abstract][Hide abstract] ABSTRACT: The recent identification of mouse mammary stem cells (MaSCs) and progenitor subpopulations has enhanced the prospect of investigating the genetic control of their lineage specification and differentiation. Here we have explored the role of the Notch pathway within the mammary epithelial hierarchy. We show that knockdown of the canonical Notch effector Cbf-1 in the MaSC-enriched population results in increased stem cell activity in vivo as well as the formation of aberrant end buds, implying a role for endogenous Notch signaling in restricting MaSC expansion. Conversely, Notch was found to be preferentially activated in the ductal luminal epithelium in vivo and promoted commitment of MaSCs exclusively along the luminal lineage. Notably, constitutive Notch signaling specifically targeted luminal progenitor cells for expansion, leading to hyperplasia and tumorigenesis. These findings reveal key roles for Notch signaling in MaSCs and luminal cell commitment and further suggest that inappropriate Notch activation promotes the self-renewal and transformation of luminal progenitor cells.